Botanical Journal of the Linnean Society, 2018, 187, 483–498. With 3 figures. Nocturnal hawkmoth and noctuid moth pollination of Habenaria limprichtii (Orchidaceae) in sub-alpine meadows of the Yulong Snow Mountain (Yunnan, China) ZHI-BIN TAO1,2, ZONG-XIN REN1,*, PETER BERNHARDT3, WEI-JIA WANG1, HUAN LIANG1,2, HAI-DONG LI1 and HONG WANG1,* 1Key Laboratory for Plant Biodiversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China 2Kunming College of Life Sciences, University of Chinese Academy of Sciences, Kunming, Yunnan 650201, China 3Department of Biology, Saint Louis University, St. Louis 63103, MO, USA Received 13 June 2017; revised 4 December 2017; accepted for publication 4 April 2018 Habenaria (Orchidaceae) is a species-rich genus, usually pollinated by members of Lepidoptera, but the pollination ecology of its sub-alpine species in the Himalayas remains under-explored. We focused on three populations of the endangered, nectar-secreting H. limprichtii on Yulong Snow Mountain (eastern Himalayas). Results showed that spurs of H. limprichtii contained 10.8 μl of nectar with 26.3% dissolved sugars at night. A hawkmoth (Deilephila elpenor subsp. lewisii) and two species of settling moths (Cucullia fraterna and Trichoplusia intermixta) were the dominant pollinators. The hawkmoths had long proboscides (26.3 mm in length), matching the spur length of the flowers, and carried one to 30 pollinaria on their eyes. In contrast, settling moths had short proboscides (15–16.4 mm) carrying fewer than nine pollinaria on their legs or thoraces. These insects may have been attracted by the floral scent dominated by two aromatic benzenoid compounds. Flowers are self-compatible, but pollinators were required to produce fruits. Hand-pollination experiments showed a high level of inbreeding depression (a low proportion of seeds with well-developed embryos in self-pollinated fruits), whereas insect-mediated (natural) rates of cross-pollination were also low (13%). This suggests that most fruits were fertilized following moth-mediated autogamy/geitonogamy. Fruit set was always higher than the rate of pollinaria removal, suggesting a higher pollination efficiency due to its sectile and friable pollinia. Fruit set in natural populations was highly variable among years (28.5–93.7%), doubling in 2016. Although this is only the fourth case of moth-pollination described in Chinese Habenaria spp., it highlights the potential importance of this syndrome at higher elevations. ADDITIONAL KEYWORDS: Himalaya – pollinarium removal – reproductive success – Sphingidae. INTRODUCTION species of diurnal, settling moths for pollinia transfer (Darwin, 1862; Edens-Meier & Bernhardt, 2014). Literature on moth-pollination in Orchidaceae began Consequently, orchids pollinated by Lepidoptera in the with Darwin (1862, 1877). Historians of science allied genera Habenaria Willd., Platanthera Rich. and are well aware of Darwin’s correct prediction that Gymnadenia R.Br. remain important model systems in Angraecum sequipedale Thouars was pollinated by a modern evolutionary ecology for studying directional moth species in the family Sphingidae (Micheneau, selection (Nilsson, 1983; Schiestl & Schluter, 2009). Johnson & Fay, 2009). In fact, Darwin first addressed Habenaria is no longer regarded as monophyletic the pollination of Anacamptis (Orchis) pyramidalis (Jin et al., 2014), but with c. 800 species it is one of (L.) Rich. noting it depended on butterflies and several the most species-rich lineages of terrestrial orchids. Brazil, southern and central Africa and East Asia *Corresponding authors: E-mail: [email protected] are its centres of diversity (Dressler, 1993; Batista, and [email protected] Bianchetti & Miranda, 2006; Batista et al., 2013). © 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 187, 483–498 483 Downloaded from https://academic.oup.com/botlinnean/article-abstract/187/3/483/5037747 by Yunnan Univeristy user on 27 June 2018 484 Z.-B. TAO ET AL. There are 54 Habenaria spp. in China, 19 of which are success (Bernhardt et al., 2017). Here, we address the endemic (Chen & Cribb, 2009). With a few exceptions lack of basic information on the pollination ecology, (Thien, 1969; Singer, 2001; Suetsugu & Tanaka, 2014), fruit production and seed viability in H. limprichtii pollination by Lepidoptera appears to dominate in by asking the following questions. (1) What are the this genus, regardless of distribution. Past research pollinators of this species? Does the pollen-vector often emphasized the length of the proboscides of the spectrum include both sphingids and settling moths? insects versus dimensions of the spurs and column (2) Is H. limprichtii self-compatible? If so, does this architecture of the flowers (Smith & Snow, 1976; species experience inbreeding depression judging Nilsson & Jonsson, 1985; Singer & Cocucci, 1997; by the proportion of well-developed embryos in the Singer, 2001; Singer et al., 2007; Peter et al., 2009; seeds? (3) Does the flower emit a floral scent at night? Pedron et al., 2012; Ikeuchi, Suetsugu & Sumikawa, If so, which floral scent compounds are present? (4) 2015; Xiong, Liu & Huang, 2015; Zhang & Gao, 2017). Does this nectar-rewarding orchid show a high rate of In contrast, the floral scent of only a few Habenaria reproductive success? spp. has been analysed to date (Peter et al., 2009; Zhang & Gao, 2017), even though scent appears to be the key attractant in flowers pollinated by nocturnal MATERIAL AND METHODS moths (Raguso, 2001; Dobson, 2006). Most Habenaria spp. studied thus far are self-compatible, but they lack STUDY SPECIES automatic self-pollination mechanisms in the absence Habenaria limprichtii is found in wet grasslands, of pollen vectors (Singer et al., 2007; Pedron et al., along margins of abandoned fields or overly grazed 2012). As members of this genus offer nectar rewards grasslands on the Yulong Snow Mountain range their flowers set fruit at a higher frequency than those through Lijiang (City) in north-western Yunnan at of allied taxa pollinated by deceit (Tremblay et al., 2300–3300 m (Fig. 1A). The population density in 2005; Singer et al., 2007; Peter et al., 2009; Pedron undisturbed wet grasslands is always higher than et al., 2012; Ikeuchi et al., 2015; Xiong et al., 2015; in sites disturbed by agriculture and grazing (Ren, Zhang & Gao, 2017). unpubl. data). Each perennial herb produces a single Incomplete records kept by Fauna Sinica suggest inflorescence bearing multiple flowers. These flowers that c. 20% of all hawkmoth species (Sphingidae) are greenish-white without any noticeable scent occur in China (Zhu & Wang, 1997), but the roles of during the daytime, but they produce a strong and sphingids and settling moths (e.g. Noctuidae and pleasant fragrance at night. Distinctive characters of Geometridae) as pollinators of the Chinese flora the floral column include the great distance between remains poorly documented (Johnson et al., 2016) the two pollinium sacs (locules) in the same anther with only a few recent publications (Tian et al., 2004; (9.52 ± 0.86 mm; N = 100, mean ± SD; Fig. 1B) and the Xiong et al., 2015; Zhang & Gao, 2017). Xiong, Liu & broad rostellum lobe contains two discrete viscidia Huang (2015) found that three species of sphingid and is, in turn, flanked by two receptive stigmatic moths carried the pollinaria of Habenaria glaucifolia lobes. Therefore, each column holds two, separate Bur. & Franch. at the bases of their proboscides but pollinaria (see below). The perianth segments either they were not able to observe moth visitations until darken as these flowers age or following pollination. the final three years of their impressive eight-year Flowering periods for populations in Lijiang occur study. Reproductive success of H. glaucifolia varied from mid-July to mid-September and their mature seasonally with a maximum 46% conversion rate of fruits dehisce in October. flowers into fruit. Sphingid moths were also observed to pollinate sympatric populations of H. davidii Franch. and H. fordii Rolfe (Zhang & Gao, 2017). STUDY SITES In contrast, Habenaria limprichtii Schltr. is Our study site is a wet meadow (Yushuizhai, YSZ) distributed through south-western China into belonging to the Lijiang Forest Ecosystem Research Vietnam and northern Thailand (Chen & Cribb, 2009) Station (Lijiang Alpine Botanical Garden) and is from 100–3500 m but it is now found less frequently operated by the Kunming Institute of Botany, Chinese in forests and wet grasslands of south-western China Academy of Sciences (CAS). We selected this site for due to increasing urbanization, deforestation and hand-pollination experiments and observations of climate change (Liu et al., 2015; Ren et al., unpubl. floral foragers. It is located at 2725 m a.s.l., and the data). A future conservation programme for any station is adjacent to a tourist and cultural centre species requires dependable information on the for the Naxi people. Consequently, vegetation in the decline of its remaining populations and that may station has been disturbed occasionally by trampling include identification of factors that limit reproductive from tourists, grazing by horses and browsing by © 2018 The Linnean Society of London, Botanical Journal of the Linnean Society, 2018, 187, 483–498 Downloaded from https://academic.oup.com/botlinnean/article-abstract/187/3/483/5037747 by Yunnan Univeristy user on 27 June 2018 NOCTURNAL MOTH POLLINATION